Drug-induced change in transmitter identity is a shared mechanism generating cognitive deficits

Cognitive deficits are long-lasting consequences of drug use, yet the convergent mechanism by which classes of drugs with different pharmacological properties cause similar deficits is unclear. We find that both phencyclidine and methamphetamine, despite differing in their targets in the brain, cause the same glutamatergic neurons in the medial prefrontal cortex of male mice to gain a GABAergic phenotype and decrease expression of their glutamatergic phenotype. Suppressing drug-induced gain of GABA with RNA-interference prevents appearance of memory deficits. Stimulation of dopaminergic neurons in the ventral tegmental area is necessary and sufficient to produce this gain of GABA. Drug-induced prefrontal hyperactivity drives this change in transmitter identity. Returning prefrontal activity to baseline, chemogenetically or with clozapine, reverses the change in transmitter phenotype and rescues the associated memory deficits. This work reveals a shared and reversible mechanism that regulates the appearance of cognitive deficits upon exposure to different drugs.

We then examined the expression of the GABA vesicular transporter (VGAT) and VGLUT1 in mCherry + neurons that co-expressed or gained GABA. We used fluorescent in situ hybridization (FISH) to detect transcripts for mCherry, for the GABA synthetic enzyme (GAD1), and for either VGAT or VGLUT1.
To reveal changes in expression levels, we selectively decreased the amplification for VGAT and VGLUT1 to obtain punctate staining (Fig. 1, E and F). In the PL of PCP-treated mice, the density of neurons expressing both mCherry and GAD1 (mCherry + /GAD1 + ) was 1.6-fold higher than in controls ( fig. S3F), mirroring the PCP-induced increase in number of mCherry + /GAD67 + neurons (Fig. 1C). Across treatments, mCherry + /GAD1 + neurons expressed VGAT at the level of GABAergic neurons (labeled with GAD1 and not mCherry) ( Fig. 1G and fig. S3, A, B and D). At the same time, the expression level of VGLUT1 in mCherry + /GAD1 + neurons decreased by ~55% compared to that of glutamatergic cells expressing mCherry and not GAD1 in PCP-treated mice (Fig. 1H) and by 46% in saline controls (fig. S3, C and E). Thus, both the glutamatergic neurons that gained GABA after PCP-exposure, as well as those expressing GABA in drug-naïve conditions, are characterized by high expression levels of VGAT and lowered expression levels of VGLUT1.
We next asked whether PCP-treatment affects the transmitter phenotype of PL GABAergic neurons. No difference was observed in the number of neurons expressing GABA and not mCherry between PCP-treated animals and controls (8594±340 vs 8837±271) (fig. S1, E and F). However, PCP and other NMDA receptor antagonists have been shown to reduce expression of GAD67 and parvalbumin in prefrontal cortex parvalbumin-positive (PV + ) interneurons (16,17). Because variability in the number of GABAergic neurons scored could have prevented the detection of loss of GABA from a small number of PV + neurons, we quantified the number of PV + neurons expressing GAD67 after PCP-treatment by permanently labeling them with a PV CRE ::TdTomato mouse line. PCP caused 223±45 TdTomato + neurons to stop expressing GAD67 ( fig. S4). This result suggests that, except for its effect on PV + neurons, PCP does not affect the GABAergic phenotype of PL interneurons.
To investigate whether glutamatergic neurons that have gained GABA contribute to cognitive deficits, we selectively suppressed GABA expression in PL glutamatergic neurons by injecting a Credependent adeno-associated virus (AAV) expressing shRNA for GAD1 (AAV-DIO-shGAD1-GFP or AAV-DIO-shScr-GFP as control) in the PL of VGLUT1 CRE mice before exposure to PCP (Fig. 1, I and J). shGAD1 suppressed GABA expression in transfected cells ( fig. S5, A to C), and reduced the number of PL neurons co-expressing GAD1 and VGLUT1 transcripts in both PCP-and saline-treated mice to half of that in saline-ShScr controls (Fig. 1K). Having efficiently overridden PCP-induced gain of GABA, we examined the impact of shGAD1 on PCP-induced behavior. While shGAD1 did not affect PCP-induced hyperlocomotion on the first day of treatment, it prevented appearance of locomotor sensitization after a 10-day PCP-exposure ( Fig.   1L and fig. S5, D and E), indicating that PCP-induced gain of GABA is required for sensitization to the acute locomotor effect of the drug. We next focused on deficits in recognition and working memory, since these behaviors are affected by repeated exposure to PCP (13,18) and are regulated by the PL (19,20). shGAD1 prevented PCP-induced impairments of recognition memory in the novel object recognition test (NORT) (Fig. 1M) and deficits in spatial working memory in the spontaneous alternation task (SAT) (Fig. 1N). Neither Glutamatergic neurons that co-express GABA or gain it after treatment with either drug were most prevalent in layer 2/3 and layer 5 of the PL (Fig. 1D, Fig. 2C, and fig. S7). These PL layers innervate the nucleus accumbens (NAc) (23), which modulates behaviors that are affected by repeated intake of PCP or METH (24)(25)(26). To determine whether neurons that switch transmitter identity project to the NAc, we injected fluoro-gold (FG) into the NAc of VGLUT1 CRE ::mCherry mice, treated them with PCP, METH or saline, and screened the PL for mCherry + /GABA + neurons expressing the retrograde tracer ( fig. S8, A to C). In both PCP-and METH-treated mice, ~0.9% of FG + neurons were mCherry + /GABA + . Such cells were less frequent PCP+PCP; P+M: PCP+METH). (K) Experimental protocol to learn whether serial administration of PCP and METH changes the transmitter phenotype of the same number of neurons as PCP alone, causes neurons that have gained GAD1 to lose it, or enables other neurons to gain GAD1. (L) Quantification of these neurons in mice treated as described in (K) (n=4 to 5 mice per group). Scale bar, (G) 20 µm. Statistical significance (**P<0.01, ****P<0.0001) was assessed using unpaired t-test or Mann Whitney test (B), Kruskal-Wallis followed by Dunn's test (D and E), and two-way ANOVA followed by Tukey's test (I and L). Data are mean ± SEM. Further statistical details are presented in Table S2.
in controls (~0.3% of the total number of FG + neurons) ( fig. S8D), indicating that neurons changing transmitter identity with drug-treatment project to the NAc.
Since both PCP and METH affect the transmitter phenotype of PL glutamatergic neurons that have the NAc as a shared downstream target, we asked whether both drugs change the transmitter identity of the same cells. If PCP and METH changed the transmitter identity of different cells, administering the two drugs one after the other should induce gain of GABA in neurons that have not gained it after treatment with the first drug. To determine whether this was the case, we genetically labeled neurons expressing GABAergic markers during the interval between the delivery of PCP and METH, using VGAT FLP ::CreER T ::TdTomato cON/fON mice in which neurons expressing VGAT at the time of tamoxifen administration are permanently labeled with TdTomato (Fig. 2F). We first injected tamoxifen in salinetreated controls and determined that TdTomato labels neurons co-expressing VGLUT1 and GAD1 in drugnaïve conditions with 77% efficiency and 79% specificity (Fig. 2G, and fig. S9).
We then used this labelling approach to distinguish neurons expressing GAD1 in drug-naïve mice from those gaining it upon drug-exposure, by administering mice with PCP after saline-and tamoxifentreatment (Fig. 2H). PCP administration increased the total number of PL VGLUT1 + /GAD1 + neurons 2-fold compared to controls (1188±23, saline+PCP; 582±27, saline+saline) (Fig. 2I), in line with previous findings (Fig. 1K). We detected no differences in the number of VGLUT1 + /GAD1 + /TdTomato + neurons (441±43, saline+PCP; 447±34, saline+saline) and VGLUT1 + /TdTomato + neurons (99±61, saline+PCP; 120±8, saline+saline) between saline+PCP mice and saline+saline controls (Fig. 2, G to J). Changes in these numbers would have indicated that drug-treatment caused some glutamatergic neurons co-expressing GAD1 in drug-naïve conditions to lose expression of GAD1. The results indicate that PCP induces expression of GAD1 in a population of PL neurons that were not previously expressing it, without affecting the transmitter phenotype of cells co-expressing GAD1 and VGLUT1 in drug-naïve conditions. We next used VGAT FLP ::CreER T ::TdTomato cON/fON mice to determine if PCP and METH cause the same neurons to switch transmitter phenotype. Mice were treated first with PCP followed by tamoxifen administration, and then treated with either saline, METH or PCP (Fig. 2K). Across treatment groups the total number VGLUT1 + /GAD1 + neurons was unchanged (1169±46, PCP+saline; 1273±69, PCP+PCP;

Drug-induced prelimbic hyperactivity mediates the change in transmitter phenotype and linked cognitive deficits
Demonstration that both PCP and METH have the same effect on the transmitter phenotype of the same PL glutamatergic neurons prompted investigation of the underlying mechanism of drug action.
Increased neuronal activity can cause neurons to change the transmitter they express (8,27,28). Could PCP and METH induce alterations in PL activity that mediate the switch in PL neuron transmitter phenotype? Both PCP and METH increased c-fos expression in PL glutamatergic neurons by 3.8-and 3.5fold after a single injection and by 2.6-and 3.7-fold throughout a 10-day treatment ( fig. S10, A to D, and F and G). This PL hyperactivity was still present 2 days after the end of drug-treatment (fig. S10, I to K). To determine whether this increase in activity promoted the switch in transmitter phenotype, we tested whether suppression of PL hyperactivity would prevent glutamatergic neurons from gaining GABA.
Glutamatergic cells in the PL receive perisomatic inhibition from local PV + interneurons, which do not show changes in c-fos expression after administration of PCP or METH ( fig. S10, E and H). We hypothesized that chemogenetic activation of PV + neurons would suppress drug-induced hyperactivity of glutamatergic cells (29,30). To test this idea, we expressed the chemogenetic receptor PSAML-5HT3HC in mPFC PV + neurons and administered PSEM 308 immediately before acute injection of either PCP or METH We then combined chemogenetic activation of PL PV + interneurons with either PCP-or METHadministration for the duration of drug-treatment (Fig. 3, A and B). The number of VGLUT1 + /GAD1 + neurons in the PL of PCP-and METH-treated mice that received PSEM 308 was half of that of mice that did not (586±18 and 611±23 vs 1262±66 and 1222±12) and was indistinguishable from that of saline-treated controls (Fig. 3, C and D). Thus, suppression of drug-induced PL hyperactivity is sufficient to prevent glutamatergic neurons from switching their transmitter identity, indicating that hyperactivity mediates the change in transmitter phenotype.
We now tested whether blocking the change in transmitter phenotype through chemogenetic activation of PV + neurons was sufficient to prevent drug-induced cognitive deficits. In mice treated with PSEM 308 , drug-induced hyperlocomotion was absent on both the first and last days of treatment (fig. S13, A to C, and fig. S14, A to C), consistent with suppression of acute drug-induced hyperactivity of PL glutamatergic neurons (31). Suppressing PL activity prevented both PCP-and METH-induced appearance of memory deficits in both the NORT and the SAT (Fig. 3, E to H, fig. S13, F and G and fig. S14, F and G), without influencing exploratory behaviors ( fig. S13, D and E, and fig. S14, D and E). The results indicate that chemogenetic activation of PV + neurons in the PL has no effect on the behavior of saline controls and specifically affects the performance of drug-treated mice.  Table S3.   GABAergic phenotype for at least 11 days of drug washout (Fig. 3D, and PCP+saline group in Fig. 2L). As PCP-and METH-induced memory deficits are also long-lasting (13,32), we asked whether the persistence of behavioral deficits is linked to retention of the GABAergic phenotype and whether both can be reversed.

Normalizing prelimbic neuron activity after drug-exposure reverses the change in transmitter identity and the associated behavioral alterations
Clozapine, a powerful antipsychotic drug, reverses PCP-induced deficits in the NORT (13), leading us to investigate whether it also reverses the change in transmitter phenotype. VGLUT1 CRE ::mCherry mice that received PCP displayed 1.94 fold more mCherry+/GABA+ PL neurons than controls, 17 days after the end of PCP-treatment, indicating that neurons had maintained the newly acquired GABAergic phenotype ( fig. S15, A to C). In mice that received clozapine treatment after PCP, the number of mCherry + /GABA + neurons was reduced compared to that of mice treated with PCP alone (559±55 vs 1124±94) and not different from that of saline-treated controls ( fig. S15, A to C). Clozapine did not affect the number of mCherry + /GABA + neurons in saline-treated mice, suggesting that this drug selectively reverses the PCPinduced change in glutamatergic neuron transmitter identity. We confirmed that clozapine rescued PCPinduced memory deficits in the NORT and SAT, without affecting the behavioral performance of controls (fig. S15, D to J).
We next investigated the mechanisms underlying clozapine-induced reversal of the switch in transmitter identity. Because clozapine suppresses the acute PCP-induced increase in PL c-fos expression (33,34), we asked whether reversal of the gain of GABA depends on neuronal activity. After drug-treatment, the number of c-fos + glutamatergic neurons was 2.1-and 2.8-fold higher in PCP-and METH-treated mice compared to controls (fig. S10, I to K). Administration of clozapine after PCP-treatment returned c-fos expression to baseline ( fig. S16), suggesting that PL hyperactivity during drug-washout is necessary to maintain the newly acquired transmitter phenotype. If this were the case, suppressing PL hyperactivity after the transmitter switch has occurred should reverse the change. To test this hypothesis, we chemogenetically activated PV + neurons for 10 days to normalize c-fos expression in the PL of PCP-and METH-treated mice after the change in transmitter phenotype had taken place (fig. S17). More than 3 weeks after the end of drug-treatment, glutamatergic neurons in the PL of both PCP and METH-treated mice still displayed the drug-induced GABAergic phenotype (Fig. 3, I to K). Normalizing PL activity decreased the number of VGLUT1 + /GAD1 + neurons in the PL of PCP and METH-treated mice to the level of controls (588±27 and 575±9 vs 1225±38 and 1071±42) (Fig. 3, I to K). Thus, PL neuronal activity maintains the transmitter switch once it has been induced. Chemogenetically activating PL PV + interneurons after the change in transmitter phenotype had occurred also rescued memory deficits in the NORT and SAT and suppressed locomotor sensitization to both PCP and METH (Fig. 3, L to O, fig. S18 and fig. S19). Overall, these data show that suppressing PL hyperactivity following drug-exposure reverses the change in transmitter phenotype and the associated behavioral alterations.  Table S4.
PCP, METH, and other addictive substances increase phasic firing of dopaminergic neurons in the ventral tegmental area (VTA) (35,36) and increase the levels of extracellular dopamine (DA) in the striatum and prefrontal cortex (37,38). Could DA signaling be a common mediator for the PCP-and METH-induced transmitter switch? To address this question, we tested whether suppressing the activity of VTA prevented the increase in the number of PL VGLUT1 + /GAD1 + neurons (Fig. 4, A to D). These results show that drug-induced increase in activity of VTA dopaminergic neurons is required for PL neurons to change their transmitter phenotype upon treatment with PCP or METH.
It remained unclear, however, whether DA signaling alone is sufficient to induce PL neurons to switch transmitter phenotype, or whether non-dopaminergic effects of PCP or METH are involved. Phasic firing of VTA neurons can be mimicked by optogenetic stimulation of VTA dopaminergic neurons (39). We asked whether repeated optogenetic stimulation of VTA dopaminergic neurons is sufficient to induce PL glutamatergic neurons to switch transmitter identity. We expressed ChR2-YFP (or YFP as control) in VTA DAT CRE neurons and implanted an optic fiber above the VTA (Fig. 4, E and F Expression of c-fos in PL glutamatergic neurons was also increased by 2.7-fold, resembling the effect of a single dose of PCP or METH ( fig. S22, A to D). We then exposed mice to 1h of VTA stimulation per day for 10 days and analyzed the transmitter phenotype of PL glutamatergic neurons (Fig. 4E). Remarkably, the number of VGLUT1 + /GAD1 + was 1.7-fold higher in ChR2-expressing mice compared to controls (Fig. 4, G   and H, and fig. S22, E to G), demonstrating that phasic firing of dopaminergic neurons in the VTA changes the transmitter phenotype of PL glutamatergic neurons. These findings establish DA signaling as a common mediator for PCP-and METH-induced gain of GABA in PL glutamatergic neurons and suggest that exposure to other addictive substances that activate the VTA could produce similar effects.

DISCUSSION
We show that a change in the transmitter identity of PL glutamatergic neurons is a shared mechanism underlying both PCP-and METH-induced cognitive deficits. Both drugs cause the same PL neurons to acquire a new transmitter phenotype characterized by expression of GABA, GAD67, and VGAT, combined with lower levels of VGLUT1. Other PL neurons show the same transmitter phenotype in drug naïve conditions, as suggested by earlier studies (40,41). This change in transmitter phenotype causes locomotor sensitization and memory deficits in the NORT and SAT, consistent with the involvement of the PL and the NAc, which receives input from PL neurons that change their transmitter identity (19,20,(24)(25)(26)42). Given the role of GABAergic long-range projections in modulating brain oscillation and synchronization (43), gain of GABA by PL neurons projecting to the NAc may contribute to the reduction of NAc firing rates and disruption of cortex-accumbens synchronization after PCP-treatment (44).
Neuronal activity mediates this drug-induced change in transmitter identity, as expected for activitydependent neurotransmitter switching (8,27,28), and is necessary to maintain the newly acquired transmitter phenotype after the end of drug-treatment. Midbrain cholinergic neurons that change transmitter identity in response to sustained exercise spontaneously revert to expression of their original transmitter within a week of cessation of the stimulus (8). In contrast, PL glutamatergic neurons maintain their GABAergic phenotype for more than 3 weeks after the end of drug-treatment and the linked cognitive deficits are long-lasting (13,32). c-fos expression in the PL increases after acute treatment with PCP or METH (33,45), as well as after one hour of phasic stimulation of VTA dopaminergic neurons, and remains elevated for at least two weeks ( fig. S16, fig. S17, and 46, 47). Suppressing drug-induced hyperactivity during or after drug-treatment, respectively, prevents or rescues the switch in transmitter phenotype and the coupled behavioral alterations, indicating that PL hyperactivity is necessary to produce and maintain these changes. Because exposure to either PCP or METH decreases expression of PV and GAD67 (17,48), impaired function of PFC PV + interneurons may contribute to PL glutamatergic neuron hyperactivity and maintenance of the newly acquired transmitter phenotype. Chronic treatment with clozapine also reduces c-fos expression in the PL of PCP-treated mice and reverses PCP-induced changes in transmitter phenotype and behavior. This effect may be mediated by increased inhibitory input to PL glutamatergic neurons (49).
DA signaling is necessary for PL neurons to change their transmitter identity, because suppression of dopaminergic hyperactivity in the VTA during PCP-or METH-treatment prevents it. Optogenetic stimulation of phasic firing of dopaminergic neurons in the VTA is sufficient to cause PL hyperactivity and induce the switch in transmitter phenotype. Many addictive substances promote phasic firing of these dopaminergic neurons (50). Reproducing this firing pattern optogenetically enhances DA release in the NAc (51) and replicates some of the neuroplastic and behavioral effects of cocaine, including conditioned place preference and acquisition of self-administration that can lead to compulsive-like behavior (39,51). This stimulation only models some aspects of drug intake (52) and does not account for the non-dopaminergic mechanisms through which multiple drugs of abuse differentially impact brain function and behavior.
Evidence that multiple drugs of abuse acutely promote mPFC hyperactivity (53,54), and that stimulation of VTA dopaminergic neurons is sufficient to change the transmitter identity of PL neurons, raises the possibility that drugs other than METH and PCP may induce cognitive deficits by switching the transmitter phenotype of PL glutamatergic neurons.